Role of G protein coupled inwardly rectifier K+ channels (GIRK) on the neurobiology depression

353 págs.

FTY720 attenuates excitotoxicity and neuroinflammation

Background: FTY720 (fingolimod, Gilenya(TM)), a structural analog of sphingosine-1-phosphate (S1P), is the first oral drug approved for treatment the relapsing-remitting form of multiple sclerosis (MS), and its efficacy has been related to induced lymphopenia and consequent immunosuppression via modulation of S1P(1) receptors (S1P(1)R). However, due to its lipophilic nature, FTY720 crosses the blood brain barrier (BBB) and could act directly on neural cells. In this study, we investigated the effectiveness of FTY720 as a neuroprotective agent using in vitro and in vivo models of excitotoxic neuronal death and examined if FTY720 exerts a direct action on neurons, or/and an indirect modulation of inflammation-mediated neurodegeneration as a possible mechanism of neuroprotection. Methods: Primary neuronal and organotypic cortical cultures were treated with N-methyl-D-aspartic acid (NMDA) to induce excitotoxic cell death (measured by lactate dehydrogenase (LDH) assay or propidium iodide uptake, respectively). The effects of FTY720 treatment (10, 100 and 1,000 nM) on neuronal survival were examined. As an in vivo model of neuronal death and inflammation, we used intracerebroventricular (icv) administration of kainic acid (KA; 0.5 mu g/2 mu l) in Sprague-Dawley rats. FTY720 was applied icv (1 mu g/2 mu l), together with KA, plus intraperitoneally (ip; 1 mg/kg) 24 h before, and daily, until sacrifice 3 days after icv. Rats were evaluated for neurological score, neuronal loss in CA3 hippocampal region and activation of microglia at the lesion site. In addition, we tested FTY720 as a modulator of microglia responses using microglial cell cultures activated with lipopolysaccharide (LPS) and its effects in stress signalling pathways using western blotting for p38 and JNK1/2 mitogen-activated protein kinases (MAPKs). Results: FTY720 was able to reduce excitotoxic neuronal death in vitro. Moreover, in vivo repeated FTY720 administration attenuated KA-induced neurodegeneration and microgliosis at the CA3 lesion site. Furthermore, FTY720 negatively modulates p38 MAPK in LPS-activated microglia, whereas it had no effect on JNK1/2 activation. Conclusions: These data support a role for FTY720 as a neuroprotective agent against excitotoxin-induced neuronal death and as a negative modulator of neuroinflammation by targeting the p38 MAPK stress signalling pathway in microglia.

Dieta hiperlipídica materna, tecido adiposo, fígado, metabolismo de carboidratos e sinalização central de leptina na prole de camundongos C57BL/6

O consumo materno de dieta hiperlipídica saturada durante a gestação e lactação favorece o desenvolvimento obesidade e anormalidades metabólicas na prole. Este trabalho teve como objetivo testar a hipótese de que a prole proveniente de mães alimentadas com dieta hiperlipídica durante a gestação e lactação desenvolve obesidade e anormalidades metabólicas e de que essas alterações estão associadas a resistência central a leptina. As ratas grávidas da linhagem C57BL/6 (n=20) foram alimentadas com dieta standard chow (SC; 19% de lipídeos) ou dieta hiperlipídica (HF; 49% de lipídeos) durante todo período de gestação e lactação. Após o desmame, a prole de machos foi dividida em quatro grupos experimentais, de acordo com a dieta das mães e da prole: SC(mães)/SC(prole), SC/HF, HF/SC e HF/HF (n=12/gp). As características metabólicas foram avaliadas pela curva de ganho de peso; medida da pressão arterial; glicose de jejum, área sob a curva no teste oral de tolerância a glicose; concentrações de triglicerídeos hepáticos e estimativa da esteatose hepática; análise plasmática de insulina e leptina e; distribuição e análise morfológica do tecido adiposo. Para analisar a sensibilidade a leptina, os quatro grupos originais foram subdivididos em dois grupos cada (veículo ou leptina-5g) para verificar a resposta alimentar (g) após o tratamento agudo intracerebroventricular (ICV) e a sinalização hipotalâmica de leptina. A dieta HF durante o período pós-desmame (grupo SC/HF), durante gestação e lactação (grupo HF/SC), ou ambos os períodos (grupo HF/HF), promoveu aumento da massa corporal. No que concerne as alterações hepáticas e a ação da insulina, a dieta HF durante o período perinatal favoreceu 25% de esteatose hepática, hiperinsulinemia e hiperleptinemia, enquanto os demais grupos experimentais SC/HF e HF/HF, demonstraram um padrão mais exacerbado. A avaliação da distribuição e morfometria do tecido adiposo demonstra o importante papel da dieta HF perinatal em amplificar a habilidade de estocar gordura visceral na prole. Considerando a ação central da leptina nos grupos tratados, a resposta alimentar mostrou-se atenuada em SC/HF, indicando o efeito determinante da dieta pós-desmame sobre a ação da leptina nesse modelo. Os resultados indicam fortemente que a dieta HF materna afeta a saúde da prole adulta. Especificamente, a prole programada apresenta esteatose hepática, hipertrofia de adipócitos, aumento de gordura visceral, hiperleptinemia e resistência a insulina. Esse fenótipo não está associado a resistência central a leptina na prole aos três meses de idade.

Bombinakinin M gene associated peptide, a novel bioactive peptide from skin secretions of the toad Bombina maxima

A novel 28-amino acid peptide, termed bombinakinin-GAP, was purified and characterized from skin secretions of the toad Bombina maxima. Its primary structure was established as DMYEIKQYKTAHGRPPICAPGEQCPIWV-NH2, in which two cysteines form a disulfide bond. A FASTA search of SWISS-PROT databank detected a 32% sequence identity between the sequences of the peptide and a segment of rat cocaine- and amphetamine-regulated transcript (CART). Intracerebroventricular (i.c.v.) administration of the peptide induced a significant decrease in food intake in rats, suggesting that it played a role in the control of feeding by brain. Analysis of its cDNA structure revealed that this peptide is coexpressed with bombinakinin M, a bradykinin-related peptide from the same toad. Bombinakinin-GAP appears to be the first example of a novel class of bioactive peptides from amphibian skin, which may be implicated in feeding behavior. (C) 2003 Elsevier Science Inc. All rights reserved.

Cardiovascular actions of trout urotensin II in the conscious trout, Oncorhynchus mykiss

The central and peripheral cardiovascular effects of synthetic trout urotensin II (UII) were investigated in the conscious rainbow trout. Intracerebroventricular injection of 50 pmol UII produced a slight (3%) but significant (P < 0.05) increase in heart rate but had no effect on mean arterial blood pressure. Injection of 500 pmol UII icy produced a significant (P < 0.05) rise (8%) in blood pressure with no change in heart rate. In contrast to the weak presser effect of centrally administered UII, intra-arterial injection of UII produced a dose-dependent increase in arterial blood pressure and decrease in heart rate with significant (P < 0.05) effects on both parameters observed at a dose of 25 pmol. Higher doses of the peptide produced a sustained decrease in cardiac output that accompanied the bradycardia and rise in arterial blood pressure. The UII-induced bradycardia, but not the increase in pressure, was abolished by pretreatment with phentolamine. Trout UII produced a sustained and dose-dependent contraction of isolated vascular rings prepared from trout efferent branchial [-log 50% of the concentration producing maximal contraction (pD(2)) = 8.30] and celiacomesenteric (pD(2) = 8.22) arteries but was without effects on vascular rings from the anterior cardinal vein. The data indicate that the presser effect of UII in trout is mediated predominantly, if not exclusively, by an increase in systemic vascular resistance. The UII-induced hypertensive response does not seem to involve release of catecholamines, but the bradycardia may arise from adrenergic-mediated activation of cardioinhibitory baroreflexes.

Orally bioavailable small molecule drug protects memory in Alzheimer's disease models

Oligomers of beta-amyloid (Aß) are implicated in the early memory impairment seen in Alzheimer's disease before to the onset of discernable neurodegeneration. Here, the capacity of a novel orally bioavailable, central nervous system-penetrating small molecule 5-aryloxypyrimidine, SEN1500, to prevent cell-derived (7PA2 [conditioned medium] CM) Aß-induced deficits in synaptic plasticity and learned behavior was assessed. Biochemically, SEN1500 bound to Aß monomer and oligomers, produced a reduction in thioflavin-T fluorescence, and protected a neuronal cell line and primary cortical neurons exposed to synthetic soluble oligomeric Aß1-42. Electrophysiologically, SEN1500 alleviated the in vitro depression of long-term potentiation induced by both synthetic Aß1-42 and 7PA2 CM, and alleviated the in vivo depression of long-term potentiation induced by 7PA2 CM, after systemic administration. Behaviorally, oral administration of SEN1500 significantly reduced memory-related deficits in operant responding induced after intracerebroventricular injection of 7PA2 CM. SEN1500 reduced cytotoxicity, acute synaptotoxicity, and behavioral deterioration after in vitro and in vivo exposure to synthetic Aß and 7PA2 CM, and shows promise for development as a clinically viable disease-modifying Alzheimer's disease treatment. © 2013 Elsevier Inc.

Novel 5-aryloxypyrimidine SEN1576 as a candidate for the treatment of Alzheimer's disease

Prefibrillar assembly of amyloid-ß (Aß) is a major event underlying the development of neuropathology and dementia in Alzheimer's disease (AD). This study determined the neuroprotective properties of an orally bioavailable Aß synaptotoxicity inhibitor, SEN1576. Binding of SEN1576 to monomeric Aß 1–42 was measured using surface plasmon resonance. Thioflavin-T and MTT assays determined the ability of SEN1576 to block Aß 1–42-induced aggregation and reduction in cell viability, respectively. In vivo long-term potentiation (LTP) determined effects on synaptic toxicity induced by intracerebroventricular (i.c.v.) injection of cell-derived Aß oligomers. An operant behavioural schedule measured effects of oral administration following i.c.v. injection of Aß oligomers in normal rats. SEN1576 bound to monomeric Aß 1–42, protected neuronal cells exposed to Aß 1–42, reduced deficits in in vivo LTP and behaviour. SEN1576 exhibits the necessary features of a drug candidate for further development as a disease modifying treatment for the early stages of AD-like dementia.

Impact de l'haploinsuffisance du gène Sim1 sur le développement et la fonction du noyau paraventriculaire de l'hypothalamus

L’obésité provient d’un déséquilibre de l’homéostasie énergétique, c’est-à-dire une augmentation des apports caloriques et/ou une diminution des dépenses énergétiques. Plusieurs données, autant anatomiques que physiologiques, démontrent que l’hypothalamus est un régulateur critique de l’appétit et des dépenses énergétiques. En particulier, le noyau paraventriculaire (noyau PV) de l’hypothalamus intègre plusieurs signaux provenant du système nerveux central (SNC) et/ou de la périphérie, afin de contrôler l’homéostasie énergétique via des projections axonales sur les neurones pré-ganglionnaires du système autonome situé dans le troc cérébral et la moelle épinière. Plusieurs facteurs de transcription, impliqués dans le développement du noyau PV, ont été identifiés. Le facteur de transcription SIM1, qui est produit par virtuellement tous les neurones du noyau PV, est requis pour le développement du noyau PV. En effet, lors d’une étude antérieure, nous avons montré que le noyau PV ne se développe pas chez les souris homozygotes pour un allèle nul de Sim1. Ces souris meurent à la naissance, probablement à cause des anomalies du noyau PV. Par contre, les souris hétérozygotes survivent, mais développent une obésité précoce. De façon intéressante, le noyau PV des souris Sim1+/- est hypodéveloppé, contenant 24% moins de cellules. Ces données suggèrent fortement que ces anomalies du développement pourraient perturber le fonctionnement du noyau PV et contribuer au développement du phénotype d’obésité. Dans ce contexte, nous avons entrepris des travaux expérimentaux ayant pour but d’étudier l’impact de l’haploinsuffisance de Sim1 sur : 1) le développement du noyau PV et de ses projections neuronales efférentes; 2) l’homéostasie énergétique; et 3) les voies neuronales physiologiques contrôlant l’homéostasie énergétique chez les souris Sim1+/-. A cette fin, nous avons utilisé : 1) des injections stéréotaxiques combinées à des techniques d’immunohistochimie afin de déterminer l’impact de l’haploinsuffisance de Sim1 sur le développement du noyau PV et de ses projections neuronales efférentes; 2) le paradigme des apports caloriques pairés, afin de déterminer l’impact de l’haploinsuffisance de Sim1 sur l’homéostasie énergétique; et 3) une approche pharmacologique, c’est-à-dire l’administration intra- cérébroventriculaire (i.c.v.) et/ou intra-péritonéale (i.p.) de peptides anorexigènes, la mélanotane II (MTII), la leptine et la cholécystokinine (CCK), afin de déterminer l’impact de l’haploinsuffisance de Sim1 sur les voies neuronales contrôlant l’homéostasie énergétique. Dans un premier temps, nous avons constaté une diminution de 61% et de 65% de l’expression de l’ARN messager (ARNm) de l’ocytocine (Ot) et de l’arginine-vasopressine (Vp), respectivement, chez les embryons Sim1+/- de 18.5 jours (E18.5). De plus, le nombre de cellules produisant l’OT et la VP est apparu diminué de 84% et 41%, respectivement, chez les souris Sim1+/- adultes. L’analyse du marquage axonal rétrograde des efférences du noyau PV vers le tronc cérébral, en particulier ses projections sur le noyau tractus solitaire (NTS) aussi que le noyau dorsal moteur du nerf vague (X) (DMV), a permis de démontrer une diminution de 74% de ces efférences. Cependant, la composition moléculaire de ces projections neuronales reste inconnue. Nos résultats indiquent que l’haploinsuffisance de Sim1 : i) perturbe spécifiquement le développement des cellules produisant l’OT et la VP; et ii) abolit le développement d’une portion importante des projections du noyau PV sur le tronc cérébral, et notamment ses projections sur le NTS et le DMV. Ces observations soulèvent donc la possibilité que ces anomalies du développement du noyau PV contribuent au phénotype d’hyperphagie des souris Sim1+/-. En second lieu, nous avons observé que la croissance pondérale des souris Sim1+/- et des souris Sim1+/+ n’était pas significativement différente lorsque la quantité de calories présentée aux souris Sim1+/- était la même que celle consommée par les souris Sim1+/+. De plus, l’analyse qualitative et quantitative des tissus adipeux blancs et des tissus adipeux bruns n’a démontré aucune différence significative en ce qui a trait à la taille et à la masse de ces tissus chez les deux groupes. Finalement, au terme de ces expériences, les souris Sim1+/--pairées n’étaient pas différentes des souris Sim1+/+ en ce qui a trait à leur insulinémie et leur contenu en triglycérides du foie et des masses adipeuses, alors que tous ces paramètres étaient augmentés chez les souris Sim1+/- nourries ad libitum. Ces résultats laissent croire que l’hyperphagie, et non une diminution des dépenses énergétiques, est la cause principale de l’obésité des souris Sim1+/-. Par conséquent, ces résultats suggèrent que : i) l’haploinsuffisance de Sim1 est associée à une augmentation de l’apport calorique sans toutefois moduler les dépenses énergétiques; ii) l’existence d’au moins deux voies neuronales issues du noyau PV : l’une qui régule la prise alimentaire et l’autre la thermogénèse; et iii) l’haploinsuffisance de Sim1 affecte spécifiquement la voie neuronale qui régule la prise alimentaire. En dernier lieu, nous avons montré que l’injection de MTII, de leptine ainsi que de CCK induit une diminution significative de la consommation calorique des souris des deux génotypes, Sim1+/+ et Sim1+/-. De fait, la consommation calorique cumulative des souris Sim1+/- et Sim1+/+ est diminuée de 37% et de 51%, respectivement, durant les 4 heures suivant l’administration i.p. de MTII comparativement à l’administration d’une solution saline. Lors de l’administration i.c.v. de la leptine, la consommation calorique cumulative des souris Sim1+/- et Sim1+/+ est diminuée de 47% et de 32%, respectivement. Finalement, l’injection i.p. de CCK diminue la consommation calorique des souris Sim1+/- et Sim1+/+ de 52% et de 36%, respectivement. L’ensemble des résultats suggère ici que l’haploinsuffisance de Sim1 diminue l’activité de certaines voies neuronales régulant l’homéostasie énergétique, et particulièrement de celles qui contrôlent la prise alimentaire. En résumé, ces travaux ont montré que l’haploinsuffisance de Sim1 affecte plusieurs processus du développement au sein du noyau PV. Ces anomalies du développement peuvent conduire à des dysfonctions de certains processus physiologiques distincts régulés par le noyau PV, et notamment de la prise alimentaire, et contribuer ainsi au phénotype d’obésité. Les souris hétérozygotes pour le gène Sim1 représentent donc un modèle animal unique, où l’hyperphagie, et non les dépenses énergétiques, est la principale cause de l’obésité. En conséquence, ces souris pourraient représenter un modèle expérimental intéressant pour l’étude des mécanismes cellulaires et moléculaires en contrôle de la prise alimentaire.

Subchronic 3, 4-methylenedioximethamphetamine (mdma) reverts anxiety induced by chronic mild stress

Here we report the effects of subchronic 3, 4-Methylenedioximethamphetamine (MDMA) on the elevated plus-maze, a widely used animal model of anxiety. Rats exposed to a mild chronic stress (MCS) protocol received intracerebroventricular microinjections of the selective serotonin reuptake inhibitor (SSRI) – fluoxetine (2.0ug/ul) or 3, 4-Methylenedioximethamphetamine (MDMA, 2.0ug/ul) for seven days. On the eighth day rats were tested in the elevated plus-maze. Our results showed that sub-chronic MDMA interacted with MCS leading to a decrease in anxiety-related behaviors including: percentage of open arms entries (F[2,26]=4.00; P=0.031), time spent in the open arms (F[2,26]=3.656; P=0.040) and time spent in the open arms extremities (F[2,26]=5.842; P=0.008). These results suggest a potential effect of MDMA in the reversion of the emotional significance of aversive stimuli.

Hypothalamic Actions of Tumor Necrosis Factor alpha Provide the Thermogenic Core for the Wastage Syndrome in Cachexia

TNF alpha is an important mediator of catabolism in cachexia. Most of its effects have been characterized in peripheral tissues, such as skeletal muscle and fat. However, by acting directly in the hypothalamus, TNF alpha can activate thermogenesis and modulate food intake. Here we show that high concentration TNF alpha in the hypothalamus leads to increased O(2) consumption/CO(2) production, increased body temperature, and reduced caloric intake, resulting in loss of body mass. Most of the thermogenic response is produced by beta 3-adrenergic signaling to the brown adipose tissue (BAT), leading to increased BAT relative mass, reduction in BAT lipid quantity, and increased BAT mitochondria density. The expression of proteins involved in BAT thermogenesis, such as beta 3-adrenergic receptor, peroxisomal proliferator-activated receptor-gamma coactivator-1 alpha, and uncoupling protein-1, are increased. In the hypothalamus, TNF alpha produces reductions in neuropeptide Y, agouti gene-related peptide, proopiomelanocortin, and melanin-concentrating hormone, and increases CRH and TRH. The activity of the AMP-activated protein kinase signaling pathway is also decreased in the hypothalamus of TNF alpha-treated rats. Upon intracerebroventricular infliximab treatment, tumor-bearing and septic rats present a significantly increased survival. In addition, the systemic inhibition of beta 3-adrenergic signaling results in a reduced body mass loss and increased survival in septic rats. These data suggest hypothalamic TNF alpha action to be important mediator of the wastage syndrome in cachexia. (Endocrinology 151: 683-694, 2010)

The Central Nervous System as Target for Antihypertensive Actions of a Proline-Rich Peptide from Bothrops jararaca Venom

Pyroglutamyl proline-rich oligopeptides, present in the venom of the pit viper Bothrops jararaca (Bj-PROs), are the first described naturally occurring inhibitors of the angiotensin I-converting enzyme (ACE). The inhibition of ACE by the decapeptide Bj-PRO-10c (intracerebroventricular injection of Bj-PRO-10c induced a significant reduction of mean arterial pressure (MAP) together with a decrease of heart rate (HR) in spontaneously hypertensive rats, indicating that Bj-PRO-10c may act on the central nervous system. In agreement with its supposed neuronal action, this peptide dose-dependently evoked elevations of intracellular calcium concentration ([Ca(2+)](i)) in primary culture from postnatal rat brain. The N-terminal sequence of the peptide was not essential for induction of calcium fluxes, while any changes of C-terminal Pro or Ile residues affected Bj-PRO-10c`s activity. Using calcium imaging by confocal microscopy and fluorescence imaging plate reader analysis, we have characterized Bj-PRO-10c-induced [Ca(2+)](i) transients in rat brain cells as being independent from bradykinin-mediated effects and ACE inhibition. Bj-PRO-10c induced pertussis toxin-sensitive G(i/o)-protein activity mediated through a yet unknown receptor, influx and liberation of calcium from intracellular stores, as well as reduction of intracellular cAMP levels. Bj-PRO-10c promoted glutamate and GABA release that may be responsible for its antihypertensive activity and its effect on HR. (C) 2010 International Society for Advancement of Cytometry

Inflammation of the Hypothalamus Leads to Defective Pancreatic Islet Function

Type 2 diabetes mellitus results from the complex association of insulin resistance and pancreatic beta-cell failure. Obesity is the main risk factor for type 2 diabetes mellitus, and recent studies have shown that, in diet-induced obesity, the hypothalamus becomes inflamed and dysfunctional, resulting in the loss of the perfect coupling between caloric intake and energy expenditure. Because pancreatic beta-cell function is, in part, under the control of the autonomic nervous system, we evaluated the role of hypothalamic inflammation in pancreatic islet function. In diet-induced obesity, the earliest markers of hypothalamic inflammation are present at 8 weeks after the beginning of the high fat diet; similarly, the loss of the first phase of insulin secretion is detected at the same time point and is restored following sympathectomy. Intracerebroventricular injection of a low dose of tumor necrosis factor a leads to a dysfunctional increase in insulin secretion and activates the expression of a number of markers of apoptosis in pancreatic islets. In addition, the injection of stearic acid intracerebroventricularly, which leads to hypothalamic inflammation through the activation of tau-like receptor-4 and endoplasmic reticulum stress, produces an impairment of insulin secretion, accompanied by increased expression of markers of apoptosis. The defective insulin secretion, in this case, is partially dependent on sympathetic signal-induced peroxisome proliferator receptor-gamma coactivator Delta a and uncoupling protein-2 expression and is restored after sympathectomy or following PGC1 alpha expression inhibition by an antisense oligonucleotide. Thus, the autonomic signals generated in concert with hypothalamic inflammation can impair pancreatic islet function, a phenomenon that may explain the early link between obesity and defective insulin secretion.

Role of brain nitric oxide in the thermoregulation of broiler chicks

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)